Method for producing porcine epidemic diarrhea virus antigen by paper carrier method

Abstract

The invention discloses a method for producing a porcine epidemic diarrhea virus antigen by a paper carrier method, and belongs to the technical field of preparation of vaccines for pigs. The method comprises the following steps: resuscitating Vero cells, gradually propagating by using a T75 square bottle, a T225 square bottle and a 15L spinner bottle, soaking and washing a paper carrier by using water for injection at 80 DEG C or above, soaking and sterilizing by using PBS (Phosphate Buffer Solution) with the pH value of 7.4, cleaning by using a low-serum culture medium, pre-culturing the paper carrier and the culture medium in a 10L bioreactor, performing perfusion culture for 5 days after the cells enter a tank, inoculating viruses according to MOI0.1, and harvesting antigens twice. According to the method, the antigen titer is high, the labor cost is reduced, the operation is simplified, and the method can be used for preparing high-quality porcine epidemic diarrhea vaccines.

Description

Technical Field

[0001] This invention relates to the field of swine vaccine preparation technology, specifically to a method for producing porcine epidemic diarrhea virus antigen using a paper carrier method. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a major disease causing mortality in newborn piglets, resulting in significant economic losses to the livestock industry in recent years. Currently, prevention and control primarily rely on vaccination. In the preparation of PEDV vaccines, PEDV antigens are mainly obtained by infecting Vero cells, culturing them, and harvesting the culture medium. Vero cells are adherent cells, and the current method of preparing vaccines using roller bottle culture suffers from low antigen titer, high labor costs, and cumbersome procedures, thus affecting the final vaccine potency and consequently, the vaccine's immunizing efficacy. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for producing porcine epidemic diarrhea virus antigen using a paper carrier method, specifically achieved through the following technical solution: A method for producing porcine epidemic diarrhea virus antigen using a paper disc carrier method includes the following steps: Step 1: Culture adherent cells in square bottles and roller bottles; take frozen Vero cells, thaw them, and then propagate them sequentially in T225 square bottles and 15L roller bottles. Step 2, Pretreatment of paper disc carriers: First, soak in water for injection at 80°C or higher, then rinse with water for injection, then soak and clean with PBS solution and sterilize, and then clean with 199 low serum medium. Step 3, Pre-culture; Add pretreated paper carriers and culture medium to the bioreactor and pre-culture for 24 hours; Step 4, cell loading: Digest the cells cultured in the 15L roller flask in Step 1 and determine the total cell count; 4 hours before loading the cells into the reactor, remove the old culture medium, add new cell culture medium, keep the temperature constant to 37℃, and maintain pH balance while stirring. Then dilute the cells and add them to the reactor. Step 5, perfusion culture; Step 6, inoculation: Discharge the culture medium from the bioreactor into the waste tank, add an equal amount of culture medium to the reactor, inoculate with the virus at an MOI of 0.1, and add the seed virus to the reactor; after the virus has been adsorbed for 6 hours, discharge all the culture medium from the reactor, add an equal amount of fresh culture medium, and continue culturing. Step 7: After culturing for 13 to 18 hours, harvest all the culture medium in the reactor and then add new culture medium; Step 8: After culturing for another 24 hours, harvest all the culture medium from the reactor.

[0004] The specific operation of step 1 is as follows: take the frozen Vero cells, thaw them and culture them in a T75 square bottle, pass the cells after 3 days, and then expand them to a T225 square bottle and a 15L roller bottle.

[0005] The pH of the PBS solution is 7.4.

[0006] The specific operation of step 3 is as follows: a bioreactor with a culture volume of 10L is used, 270g of paper carrier is used, 10L of culture medium is added, and pre-culture is carried out for 24 hours. The reactor parameters are set as follows: rotation speed 100rpm, pH 7.2, temperature 37℃, dissolved oxygen 60%.

[0007] In step 4, the total cell count should be greater than 5E+09 cells; the total cell count after dilution is between 3E+09 cells and 4E+09 cells.

[0008] In step 4, after adding new cell culture medium, the temperature is kept constant at 37°C and the pH value reaches 7.1. The reactor speed is adjusted to 120 rpm. After adding the diluted cell suspension for 5 minutes, the reactor speed is adjusted from 120 rpm to 70 rpm. After the solution in the tank becomes clear, the speed is adjusted to 100 rpm for culture.

[0009] The specific operation of step 5 is as follows: no perfusion on the first day; perfusion volume of 6L on the second day, with inflow and outflow at 10rpm; perfusion volume of 13L on the third day, with inflow and outflow at 20rpm; perfusion volume of 30L on the fourth day, with inflow and outflow at 40rpm; and perfusion volume of 50L on the fifth day, with inflow and outflow at 65 to 70rpm.

[0010] In step 2, the number of times the water for injection is used for rinsing is 5.

[0011] The technical solution of this invention has the following advantages: The paper-carrier method for producing porcine epidemic diarrhea virus antigen described in this invention involves first progressively propagating and cryopreserving Vero cells in square bottles and roller bottles to ensure a sufficient supply of viable cells. Then, the paper carriers undergo standardized pretreatment processes including soaking in water for injection at temperatures above 80°C, multiple rinsing, sterilization with PBS, and washing with low-serum culture medium. Subsequently, precise control of pre-culture parameters within a bioreactor, combined with cell number regulation during cell loading, dynamic adjustment of rotation speed, and staged perfusion culture, creates a stable and suitable environment for Vero cell adhesion and porcine epidemic diarrhea virus proliferation. This significantly improves antigen titer and effectively solves the problem of low antigen titer leading to poor vaccine efficacy in traditional methods. This addresses the issue of low antigen titer in the final vaccine product. Furthermore, the entire production process, from cell propagation and carrier processing to culture, inoculation, and harvesting, is systematically conducted within a bioreactor, reducing the cumbersome manual transfers and operations required in traditional roller bottle culture, lowering labor costs, and avoiding interference from variations in manual operation on culture conditions. In addition, the appropriate timing of inoculation and the two-harvest process design further improve antigen production efficiency, ultimately providing high-quality antigen raw materials for the preparation of porcine epidemic diarrhea vaccine, ensuring vaccine immunization efficacy. This comprehensively overcomes the technical shortcomings of traditional roller bottle culture methods in the background technology, such as low antigen titer, high labor costs, cumbersome operations, and compromised vaccine immunization efficacy. Attached Figure Description

[0012] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0013] Figure 1 The above are post-harvest antigen titer diagrams for Examples 1 to 3 of the present invention. Detailed Implementation

[0014] The technical solution of the present invention will be clearly and completely described below through embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0016] This invention provides a method for culturing Vero cells using a paper disc carrier method in a bioreactor and preparing CV777 antigen by inoculating Vero cells with CV777 virus, which results in higher antigen titer, saves tedious manual operations, and increases production efficiency.

[0017] The invention will now be described in detail through specific implementation batches.

[0018] Example 1 (Batch 1) 1. Culture of adherent cells in square bottles and roller bottles Frozen Vero cells were taken, thawed, and cultured in a T75 square flask. After 3 days, the cells were passaged and then propagated to T225 square flasks and 15L roller bottles.

[0019] 2. Pretreatment of paper carrier Soak in water for injection at a temperature above 80°C until it is fully wetted and swelled. Rinse five times with water for injection. Soak and wash once with PBS solution at pH 7.4; Load into a bioreactor and sterilize for at least 30 minutes; After cooling, drain the PBS and wash twice with 199% low serum medium (the same below).

[0020] 3. Pre-culture A 10L bioreactor was used, with 270g of paper carrier added and 10L of culture medium added. The culture was pre-cultured for 24 hours. The reactor parameters were set as follows: rotation speed 100rpm, pH 7.2, temperature 37℃, and dissolved oxygen 60%.

[0021] 4. Cells are placed into the container. After digesting the cells cultured in the 15L roller bottle in step 1, shake the cell bottle to ensure that the cells are in full contact with the trypsin. Once a misty appearance is achieved, discard the trypsin. After the cells begin to detach and disperse, resuspend the digested cells in culture medium to prepare a cell suspension. Pour the suspension into a 5L filament bottle and count the cells using a cell counter to determine the cell density. The total number of cells should be greater than 5E+09 cells. Four hours before loading the cells into the reactor, remove the old culture medium from the pre-cultured culture medium, add new cell culture medium, keep the temperature constant to 37°C, and bring the pH to equilibrium (7.1). Then, adjust the reactor speed to 120 rpm. Based on the cell density, a cell suspension with a total cell count of 4E+09 was added to the reactor. After 5 minutes, the reactor speed was adjusted from 120 rpm to 70 rpm. After 4 hours, the solution in the tank was observed to see if it changed from turbid to clear. After it became clear, the speed was adjusted to 100 rpm for culturing. 5. Perfusion culture The culture was perfused for 5 days, and the flow rate during perfusion is shown in the table below.

[0022]

[0023] 6. Receiving poison Discharge the culture medium from the tank into the waste container, add 10L of culture medium to the reactor, and inoculate the virus according to the MOI of 0.1, adding the seed virus to the reactor. After the virus has been adsorbed for 6 hours, discharge all the culture medium from the reactor, add 10L of fresh culture medium, and continue culturing.

[0024] 7. First Harvest After 18 hours of cultivation, all the culture medium in the reactor was harvested, and then new culture medium was added. 8. Second Harvest After culturing for another 24 hours, all the culture medium in the reactor was harvested.

[0025] 9. Detect the antigen content of porcine epidemic diarrhea virus in the first and second harvests.

[0026] Example 2 (Batch 2) 1. Culture adherent cells in square bottles and roller bottles: Take frozen Vero cells, thaw them, and culture them in a T75 square bottle. After 3 days, passage the cells and then expand them to T225 square bottles and 15L roller bottles.

[0027] 2. Pretreatment of paper carrier Soak the sample three times in water for injection at a temperature of 80°C or higher to ensure it is fully saturated and swelled; then rinse it five times with water for injection. Soak and wash 1-2 times with PBS solution at pH 7.4; Load into the bioreactor and sterilize for at least 30 minutes; after cooling, drain the PBS and wash twice with 199 low serum medium (the same below).

[0028] 3. Pre-culture A 10L bioreactor was used, with 270g of paper carrier added and 10L of culture medium added. The culture was pre-cultured for 24 hours. The reactor parameters were set as follows: rotation speed 100rpm, pH 7.2, temperature 37℃, dissolved oxygen 60%.

[0029] 4. Cells are placed into the container. After digesting the cells cultured in the 15L roller bottle in step 1, shake the cell bottle to ensure that the cells are in full contact with the trypsin. Once a misty appearance is achieved, discard the trypsin. After the cells begin to detach and disperse, resuspend the digested cells in culture medium to prepare a cell suspension. Pour the suspension into a 5L filament bottle and count the cells using a cell counter to determine the cell density. The total number of cells should be greater than 5E+09 cells. Four hours before loading the cells into the reactor, remove the old culture medium from the pre-cultured culture medium, add new cell culture medium, keep the temperature constant to 37°C, and bring the pH to equilibrium (7.2). Then, adjust the reactor speed to 120 rpm. Based on the cell density, a cell suspension with a total cell count of 3E+09 was added to the reactor. After 5 minutes, the reactor speed was adjusted from 120 rpm to 70 rpm. After 1 hour, the solution in the tank was observed to see if it changed from turbid to clear. If it did not become clear, the time was appropriately delayed until it became clear. After that, the speed was adjusted to 100 rpm for culturing. 5. Irrigation culture: Irrigation culture for 5 days, with the flow rate during irrigation as shown in the table below.

[0030] 6. For virus inoculation, discharge the culture medium from the tank into the waste liquid container, add 10L of culture medium to the reactor, and inoculate the virus according to the MOI of 0.1. After the virus has been adsorbed for 6 hours, discharge all the culture medium from the reactor, add 10L of fresh culture medium, and continue culturing.

[0031] 7. After the first harvest and 15 hours of cultivation, harvest all the culture medium in the reactor and then add new culture medium; 8. After the second harvest, continue culturing for 18 hours, then harvest all the culture medium in the reactor.

[0032] 9. Test the results of the swine epidemic diarrhea virus antigen content in the first and second harvests.

[0033] Example 3 (Batch 3) 1. Adhesive adhesion in square bottles and roller bottles Cells were taken from cryopreserved Vero cells, thawed and cultured in a T75 square flask. After 3 days, the cells were passaged and then propagated to T225 square flasks and 15L roller bottles.

[0034] 2. Pretreatment of paper carrier Soak twice in water for injection at a temperature of 80°C or higher to ensure complete saturation and expansion. Rinse five times with water for injection. Soak and wash 1-2 times with PBS solution at pH 7.4; load into the bioreactor and sterilize for at least 30 minutes; After cooling, drain the PBS and wash twice with 199% low serum medium (the same below).

[0035] 3. Pre-culture: A 10L bioreactor was used, with 270g of paper carrier added to 10L of culture medium. The pre-culture was carried out for 24 hours. The reactor parameters were set as follows: rotation speed 100rpm, pH 7.2, temperature 37℃, dissolved oxygen 60%.

[0036] 4. Cells are placed into the container. After digesting the cells cultured in the 15L roller bottle in step 1, shake the cell bottle to ensure that the cells are in full contact with the trypsin. Once a misty appearance is achieved, discard the trypsin. After the cells begin to detach and disperse, resuspend the digested cells in culture medium to prepare a cell suspension. Pour the suspension into a 5L filament bottle and count the cells using a cell counter to determine the cell density. The total number of cells should be greater than 5E+09 cells. Four hours before loading the cells into the reactor, remove the old culture medium from the pre-cultured culture medium, add new cell culture medium, keep the temperature constant to 37°C, and bring the pH to equilibrium (7.2). Then, adjust the reactor speed to 120 rpm. Based on the cell density, a cell suspension with a total cell count of 3.5E+09 was added to the reactor. After 5 minutes, the reactor speed was adjusted from 120 rpm to 70 rpm. After 2.5 hours, the solution in the tank was observed to see if it changed from turbid to clear. If it did not become clear, the time was appropriately delayed until it became clear. After that, the speed was adjusted to 100 rpm for culturing. 5. Irrigation culture: Irrigation culture for 5 days, with the flow rate during irrigation as shown in the table below.

[0037] 6. For virus inoculation, discharge the culture medium from the tank into the waste liquid container, add 10L of culture medium to the reactor, and inoculate the virus according to the MOI of 0.1. After the virus has been adsorbed for 6 hours, discharge all the culture medium from the reactor, add 10L of fresh culture medium, and continue culturing.

[0038] 7. After the first harvest and 13 hours of cultivation, harvest all the culture medium in the reactor and then add new culture medium; 8. After the second harvest, continue culturing for 12 hours, then harvest all the culture medium in the reactor.

[0039] 9. Test the results of the swine epidemic diarrhea virus antigen content in the first and second harvests.

[0040] The antigen titers of the first harvest in the above batches ranged from 7.4 to 8.4 TCID50 / ml, while the antigen titers of the second harvest ranged from 7.2 to 7.9 TCID50 / ml.

[0041] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for producing porcine epidemic diarrhea virus antigen using a paper carrier method, characterized in that, Includes the following steps: Step 1: Culture adherent cells in square bottles and roller bottles; take frozen Vero cells, thaw them, and then propagate them sequentially in T225 square bottles and 15L roller bottles. Step 2, Pretreatment of paper disc carriers: First, soak in water for injection at 80°C or higher, then rinse with water for injection, then soak and clean with PBS solution and sterilize, and then clean with 199 low serum medium. Step 3, Pre-culture; Add pretreated paper carriers and culture medium to the bioreactor and pre-culture for 24 hours; Step 4, cell loading: Digest the cells cultured in the 15L roller flask in Step 1 and determine the total cell count; 4 hours before loading the cells into the reactor, remove the old culture medium, add new cell culture medium, keep the temperature constant to 37℃, and maintain pH balance while stirring. Then dilute the cells and add them to the reactor. Step 5, perfusion culture; Step 6, inoculation: Discharge the culture medium from the bioreactor into the waste liquid tank, add an equal amount of culture medium into the reactor, inoculate with the seed virus according to MOI 0.1, and add the seed virus into the reactor. Six hours after the virus is adsorbed, all the culture medium in the reactor is removed, and an equal amount of fresh culture medium is added for further cultivation. Step 7: After culturing for 13 to 18 hours, harvest all the culture medium in the reactor and then add new culture medium; Step 8: After culturing for another 24 hours, harvest all the culture medium from the reactor.

2. The method for producing porcine epidemic diarrhea virus antigen using the paper carrier method according to claim 1, characterized in that, The specific operation of step 1 is as follows: take the frozen Vero cells, thaw them and culture them in a T75 square bottle, pass the cells after 3 days, and then expand them to a T225 square bottle and a 15L roller bottle.

3. The method for producing porcine epidemic diarrhea virus antigen using the paper carrier method according to claim 1, characterized in that, The pH of the PBS solution is 7.

4.

4. The method for producing porcine epidemic diarrhea virus antigen using the paper carrier method according to claim 1, characterized in that, The specific operation of step 3 is as follows: a bioreactor with a culture volume of 10L is used, 270g of paper carrier is used, 10L of culture medium is added, and pre-culture is carried out for 24 hours. The reactor parameters are set as follows: rotation speed 100rpm, pH 7.2, temperature 37℃, dissolved oxygen 60%.

5. The method for producing porcine epidemic diarrhea virus antigen using the paper carrier method according to claim 1, characterized in that, In step 4, the total cell count should be greater than 5E+09 cells; the total cell count after dilution is between 3E+09 cells and 4E+09 cells.

6. The method for producing porcine epidemic diarrhea virus antigen using the paper carrier method according to claim 1 or 5, characterized in that, In step 4, after adding new cell culture medium, the temperature is kept constant at 37°C and the pH value reaches 7.

1. The reactor speed is adjusted to 120 rpm. After adding the diluted cell suspension for 5 minutes, the reactor speed is adjusted from 120 rpm to 70 rpm. After the solution in the tank becomes clear, the speed is adjusted to 100 rpm for culture.

7. The method for producing porcine epidemic diarrhea virus antigen using the paper carrier method according to claim 6, characterized in that, The specific operation of step 5 is as follows: no perfusion on the first day; perfusion volume of 6L on the second day, with inflow and outflow at 10rpm; perfusion volume of 13L on the third day, with inflow and outflow at 20rpm; perfusion volume of 30L on the fourth day, with inflow and outflow at 40rpm; and perfusion volume of 50L on the fifth day, with inflow and outflow at 65 to 70rpm.

8. The method for producing porcine epidemic diarrhea virus antigen using the paper carrier method according to claim 1, characterized in that, In step 2, the number of times the water for injection is used for rinsing is 5.

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